【作者】 吴淑燕；

【导师】 王兆钺；

【作者基本信息】 苏州大学 ， 内科血液学， 2006， 博士

【摘要】 近年来随着人类基因组序列图测定的完成,几乎所有的凝血因子、血小板膜糖蛋白以及其它有关成分的基因都已被克隆,有助于深入探索其结构与功能的关系。很多遗传性出血性疾病的基因异常已被发现,基因诊断是今后遗传性出血性疾病研究发展的一个主要方向。遗传性出血性疾病常导致严重的出血,需要特殊的诊断方法与特别的治疗措施。我们利用在血栓与止血领域的优势,深入开展遗传性出血性疾病的基础与临床研究,阐明其发病的分子机制,解决这类特殊患者的痛苦,并提高我院血栓与止血的研究水平。目的:分别对1例遗传性无纤维蛋白原血症(hereditary afibrinogenemia, HAF)和遗传性凝血因子XIII缺乏症(hereditary factor XIII deficiency , HFXIIID)进行基因分析和家系调查,阐明HAF和HFXIIID发病的分子机制;研究一种具有异常超微结构的遗传性巨血小板减少症(hereditary macrothrombocytopenia, HMTC)的形态与功能。方法:1.凝血酶法与免疫比浊法测定纤维蛋白原(Fg),提取HAF家系先证者及其家系成员外周血基因组DNA,PCR法扩增其Fg的FGA、FGB和FGG基因所有外显子和侧翼序列,DNA序列分析确定Fg的基因异常。将先证者突变序列、家系成员和50名正常人相应序列的PCR产物用限制性内切酶RsaⅠ消化,以进一步确定基因突变并排除基因多态性。2.分别用尿素溶解法和试剂盒法检测HFXIIID患者及其家系成员血浆FXIII的活性,并用火箭电泳法和酶联免疫吸附实验测定其FXIII抗原含量。PCR法扩增F13A基因的所有外显子和侧翼序列,DNA测序分析基因异常,用直接测序法和限制性内切酶(SfaNⅠ、NspⅠ)分析80名正常人相应序列的PCR产物以排除基因多态性。应用生物信息学软件对所发现的突变进行分子模建,探讨其致病的分子机制。3.采集HMTC患者外周静脉血,分离富血小板血浆,用流式细胞术结合多种抗血小板膜糖蛋白(GP)单克隆抗体,测定血小板膜GP Ib、GP IIb、GP IIIa、P选择素和CD63的表达;以常规超薄切片术、免疫电镜技术检测患者血小板及其异常超微结构的性质;用荧光分光光度法测定血小板5-羟色胺含量。结果:1. HAF先证者及其父亲的血浆Fg用Clauss法检测不到,免疫比浊法测定时,Fg含量均<0.02g/L,两者FGA基因外显子4第3108位核苷酸发生C→T纯合性改变,使Fg的Aa链第150位密码子(CAG,编码Gln)突变为终止密码TAG,即Q150X。更多还原

【Abstract】 Both hereditary FXIII deficiency and hereditary afibrinogenaemia are very rare and severe autosomal bleeding disorder with an average frequency of one case in 1~2 million in the general population. To date, owing to the rarity of these two kinds of deficiencies, both clinical and basic problems related to them such as the type and severity of bleeding symptoms, the underlying molecular defects and the actual management of bleeding episodes have not been fully characterized and continue to confront hematologists.Hereditary macrothrombocytopenia is a heterogeneous group of rare bleeding disorders characterized by prolonged bleeding time, thrombocytopenia and giant platelets. The pathogenic mechanism of some macrothrombocytopenias is still unresolved. In addition, some novel disorders were constantly discovered in the last few years. Recently, we found a patient with inherited macrothrombocytopenia, the morphology of whose platelets was very special containing many large electron opaque organelles.Part I: Hereditary afibrinogenemia associated with a novel nonsense mutation in the FGA gene.The molecular basis of hereditary afibrinogenemia is still poorly characterized. Up to date, only 33 abnormalities of genes encoding fibrinogen A alpha-chains (FGA), fibrinogen B beta-chains (FGB), and fibrinogen gamma-chains (FGG) have been identified. Here we reported the identification of the genetic defect underlying afibrinogenamia in a Chinese family. The proposita was a 17-year-old Chinese girl, born of a consanguineous marriage, and consanguinity is a recurrent feature in her family, besides her parents, her paternal grandparents also are first cousins The proposita displayed haemorrhagic manifestations, her blood clotting indices were prolonged and her coagulation factors were in normal ranges, whereas functional and immunological tests indicate absence of fibrinogen (<0.2g/l). The other family members were更多还原